research article the possible role of the novel cytokines...

Research ArticleThe Possible Role of the Novel Cytokines IL-35 and IL-37 inInflammatory Bowel Disease

Yanmei Li Yanan Wang Ying Liu Yatian Wang Xiuli Zuo Yanqing Li and Xuefeng Lu

Department of Gastroenterology Qilu Hospital Shandong University 107 Wenhua West Road Jinan 250012 China

Correspondence should be addressed to Xuefeng Lu luxfsdu163com

Received 10 June 2014 Revised 23 July 2014 Accepted 23 July 2014 Published 18 August 2014

Academic Editor Ishak O Tekin

Copyright copy 2014 Yanmei Li et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Interleukin- (IL-) 35 and IL-37 are newly discovered immune-suppressing cytokines They have been described in inflammatorydiseases such as collagen-induced arthritis and asthma However their expressions in inflammatory bowel disease (IBD) patientshave not been yet explored Our aim was to evaluate serum and inflamed mucosal levels in IBD patients In 20 ulcerative colitis(UC) patients 7 Crohnrsquos disease (CD) patients and 15 healthy subjects cytokine levels in serum were determined using ELISA andmucosal expression studies were performed by immunohistochemistry quantitative real-time PCR and Western blot The resultsshowed that serums IL-35 and IL-37 levels were significantly decreased in UC and CD patients compared with healthy subjectsThe cytokines levels correlated inversely with UC activity IL-35 was expressed in infiltrating immune cells while IL-37 in intestinalepithelial cells as well as inflammatory cells IBD patients had significantly higher Ebi3 p35 (two subunits of IL-35) and IL-37b geneexpressions IL-35 and IL-37 protein expressionswere higher in IBDpatients comparedwith controlsThe study showed that serumsIL-35 and IL-37 might be potentially novel biomarkers for IBD Intestinal IL-35 and IL-37 proteins are upregulated suggesting thatregulating the expression of the two cytokines may provide a new possible target for the treatment of IBD

1 Introduction

Inflammatory bowel disease (IBD) including Crohnrsquos disease(CD) and ulcerative colitis (UC) is a kind of chronicinflammatory disorder of the gastrointestinal tract Althoughthe etiology is not completely understood initiation andexacerbation of the inflammatory process seem to be dueto a massive local mucosal immune response [1] Analysisof immunoinflammatory pathways in the gut of patientswith UC or CD has shown that tissue damage is drivenby a complex and dynamic crosstalk between immune andnonimmune cells and that cytokines are keymediators of thisinterplay [2 3]

Interleukin- (IL-) 35 and IL-37 are newly discoveredimmune-suppressing cytokines IL-35 belongs to the IL-12family which contains IL-12 IL-23 and IL-27 It is composedof two subunits Epstein-Barr virus-induced gene 3 (Ebi3)and p35 (IL-12a) [4] IL-35 was shown to be secreted byFoxp3+CD4+CD25+ regulatory T cells (Tregs) in mice or aregulatory T cell population induced by IL-35 [5] andCD138+

plasma cells in experimental autoimmune encephalomyelitis(EAE) [6] Using experimental database mining and statis-tical analysis methods Li et al reported that IL-35 is notconstitutively expressed in human tissues but it is inducible inresponse to inflammatory stimuli [7] IL-37 also known as IL-1F7 is a newmember of the IL-1 family which shares commoncharacteristic symbolized by a similar 120573-barrel structure IL-37b is the largest isoform of the five variants and is expressedin a variety of normal tissues and tumors in humans [8] It wasfirst found in bone marrow and neutrophils were the mainplace for its synthesis It is mainly expressed in blood cellsrespiratory tract gastrointestinal tract and skin keratinocytes[9]

To investigate the possible role of IL-35 and IL-37 in theinflammatory process of IBD we aim to evaluate serum andmucosal levels in IBD patients To the best of our knowledgethis is the first study that explores expression through aquantitative real-time polymerase chain reaction (qRT-PCR)immunohistochemistry and Western blot of IL-35 and IL-37in inflamed colonic mucosa of IBD patients

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 136329 10 pageshttpdxdoiorg1011552014136329

2 Mediators of Inflammation

2 Materials and Methods

21 Subjects A total of 27 patients with definitive diagnosisof IBD were recruited at Shandong University Qilu Hospital20UC and 7CD patients were included during the periodfrom September 2013 to April 2014 Diagnosis was performedby the presence of history of abdomen pain diarrhea orblood in stool and macroscopic appearance by colonoscopyor double balloon endoscopy and biopsy compatible withIBD The following were relevant medical records genderage at diagnosis disease evolution extension extraintestinalmanifestations medical treatment and clinical course ofdisease UC activity was assessed byMayo score activity index[10] and CD activity was assessed by Crohnrsquos disease activityindex (CDAI) [11] Blood was drawn for the measurementof hemoglobin hematocrit and erythrocyte sedimentationrate (ESR) Additionally 15 noninflamed controls (medianage 48 yr 9 males6 females) were recruited among healthysubjects undergoing a colonoscopy because of screening forcolorectal cancer or polyp surveillance These subjects werefree from gastrointestinal symptoms and other inflamma-tory diseases Only subjects with both macroscopically andmicroscopically normal colonoscopy were included None ofthe healthy subjects in the study was taking any medicationsknown to affect the gastrointestinal tract or the immunesystem

22 Samples Collection A fasting blood sample was takenfrom all patients and healthy subjects It was centrifugedat 1500timesg for 20min at room temperature and the serumwas collected and stored at minus80∘C until analysis Duringendoscopy biopsies from colon or ileocecum or smallintestine were obtained from patients and healthy subjectsTwo biopsies to be used for RNA and protein assessmentwere snap-frozen in liquid nitrogen and then transferredto minus80∘C for storage until processing One biopsy wasplaced in formalin for pathology and immunohistochemicalstaining The study was performed after receiving writteninformed consent from all study subjects and the protocolwas approved by the Regional Ethical Review Board at QiluHospital Shandong University

23 Enzyme-Linked ImmunosorbentAssay Serums IL-35 andIL-37 were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Bio-Swamp)All cytokines assays were performed in duplicate and inaccordance with the manufacturersrsquo protocols

24 Immunohistochemistry Formalin-fixed and paraffin-embedded 4 120583m thick tissue slices were dewaxed and rehy-drated before antigen retrieval Microwave antigen retrievalmethod was then preformed with the slides immersed inEDTA antigen retrieval solution (ph 90) for 15minutes Afterthat 3 hydrogen peroxide (H

2O2) was added on the slides

to inhibit the endogenous peroxidase activity Nonspecificbinding was blocked by incubation with 10 normal goatserum in 37∘C pH75 for 30min Subsequently mouse anti-human IL-35 monoclonal antibody (Imgenex USA) at a

1 200 dilution and mouse anti-human IL-37 monoclonalantibody (Abcam USA) at a 1 250 dilution were appliedrespectively to the sections that were latter incubated at 4∘Covernight On the second day biotinylated antibody andstreptavidin-peroxidase reagent (Zhongshan Biotech China)were successively applied for 30min each at 37∘C Finally31015840-diaminobenzidine tetrahydrochloride (DAB) was used forvisualization and hematoxylin was added to counterstainSamples were viewed with Olympus IX81 microscope andimages were produced using DP Controller 121108 All ofthe slides were independently analyzed by two pathologists

25 RNA Isolation and Quantitative Real-Time PCR RNAwas extracted using Trizol Reagent (Takara Japan) follow-ing the manufacturerrsquos guidelines First-strand cDNA wassynthesized by Realtime PCR Master Mix Kit (TOYOBOJapan) in a volume of 10 120583L For quantitative real-time PCR(qRT-PCR) the LightCycler 40 instrument (Roche AppliedScience Germany) and the SYBR Green Realtime PCRMaster Mix Kit (TOYOBO Japan) were used according tothe protocol provided by the manufacturer The followingprimers were used Ebi3 forward 51015840-GCA GCA GAC GCCAAC GT-31015840 reverse 51015840-CCA TGG AGA ACA GCT GGACAT-31015840 p35 forward 51015840-CCT TCA CCA CTC CCA AAAC-31015840 reverse 51015840-TGT CTG GCC TTC TGG AGC AT-31015840IL-37 forward 51015840-GCT CAG GTG GGC TCC TGG AA-31015840reverse 51015840-GCT GAC CTC ACT GGG GCT CA-31015840 Humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH) wasused as internal control from parallel samples because thereference gene was stably expressed and its primers wereforward 51015840-GGT GGT CTC CTC TGA CTT CAA CAG-31015840and reverse 51015840-GTT GTT GTA GCC AAA TTC GTT GT-31015840 Melting curve analysis was used to confirm amplifica-tion specificity The quantification data were analyzed withLightCycler analysis software version 40 (Roche AppliedScienceGermany) and the relative target gene expressionwasnormalized on the basis ofGAPDH Results were expressed asan x-fold difference relative to the calibrator

26 Western Blot Analysis Frozen tissue samples were lysedin RIPA buffer (50mM Tris pH 74 150mM NaCl 1Triton X-100 1 sodium deoxycholate 01 SDS Bey-otime China) and PMSF (Beyotime China) followed bycentrifugation (12000 rpm 4∘C 20 minutes) after whichthe supernatants were stored at minus80∘C until use The pro-tein concentrations of the lysates were determined usingan enhanced BCA Protein Assay Kit (Beyotime China)Extracted proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Samplestransferred onto PVDFmembranes were treated with a 1 200dilution of mouse anti-human IL-35 monoclonal antibody(Imgenex USA) and anti-human IL-37monoclonal antibody(Abcam USA) followed by a 1 5000 dilution of sheep anti-mouse horseradish peroxidase-conjugated secondary anti-bodies (Zhongshan Biotech China) As a control rabbit anti-human GAPDH polyclonal antibody (Proteintech USA) andperoxidase-conjugated anti-rabbit IgG (H+L) (Proteintech

Mediators of Inflammation 3

Table 1 Demographic and clinical characteristics of IBD patients

Patients number SexAge at Disease

Disease extent Disease activity Current therapydiagnosis duration(years) (months)

Mayo scoreUC1 F 46 4 E2 7 MesalazineUC2 M 29 70 E3 12 Mesalazine corticosteroidsUC3 M 25 2 E3 9 Mesalazine corticosteroidsUC4 F 51 10 E3 11 Mesalazine corticosteroidsUC5 M 57 12 E1 5 MesalazineUC6 M 34 6 E2 6 MesalazineUC7 F 28 168 E2 3 MesalazineUC8 M 34 3 E3 11 Mesalazine corticosteroidsUC9 F 28 40 E2 11 Mesalazine corticosteroids and AZAUC10 M 59 7 E3 11 Mesalazine corticosteroidsUC11 M 24 1 E3 8 Mesalazine corticosteroidsUC12 F 27 1 E1 4 Mesalazine enemaUC13 M 27 1 E1 3 MesalazineUC14 M 58 2 E2 12 Mesalazine corticosteroidsUC15 M 65 2 E2 5 MesalazineUC16 F 15 2 E2 11 Mesalazine corticosteroidsUC17 M 22 12 E3 6 MesalazineUC18 M 37 1 E3 3 MesalazineUC19 F 63 1 E3 8 MesalazineUC20 F 28 5 E3 7 Mesalazine enemaAverage (SEM) 3785 (348) 1750 (876) 765 (072)

CDAI scoreCD1 M 60 2 L1 7 Mesalazine corticosteroids and AZACD2 M 39 180 L1 6 Mesalazine AZACD3 M 28 5 L2 8 Mesalazine corticosteroidsCD4 F 26 1 L3 9 Mesalazine corticosteroidsCD5 M 49 60 L4 9 Mesalazine corticosteroidsCD6 M 50 3 L3 6 MesalazineCD7 M 22 6 L1 7 Mesalazine corticosteroidsAverage (SEM) 3915 (544) 3671 (2518) 743 (048)UC ulcerative colitis CD Crohnrsquos disease E1 proctitis E2 leftsided colitis E3 pancolitis L1 ileal L2 colonic L3 ileocolonic L4 upper GI involvementAZA azathioprine

USA) were used to normalize Immunoreactivity was visu-alized with the ImmobilonWestern Chemiluminescent HRPSubstrate (Millipore USA) and quantified using Image Jsoftware (httprsbwebnihgovijindexhtml)

27 Statistical Analysis Statistical evaluations were per-formed with GraphPad Prism 50 (GraphPad Software IncSan Diego CA USA) and SPSS190 (SPSS Inc Chicago ILUSA) Data were expressed as mean plusmn standard error (SEM)The Mann-Whitney test was used to evaluate differencesof cytokine expression in the serum One-way analysis ofvariance followed by post hoc 119905 tests with Newman-Keulstest for multiple comparisons was used to compare the 3groups Pearson correlationwas used to calculate correlationsbetween serum cytokines levels and Mayo score One-wayanalysis of variance followed by post hoc 119905 tests with Tukey

correction formultiple comparisons was used to compare the3 groups in differences of mucosal expression In all tests a 119875value lt 005 was considered significant

3 Results

31 Demographic and Clinical Characteristics of IBD PatientsThey are described in Table 1TheMontreal classification wasused to define the extent of UC 50 had pancolitis (E3)35 had left-sided colitis (E2) and 15 had proctitis (E1)According to Mayo score six UC patients (30) showedmild activity seven (35) moderate activity and seven (35)severe activity Six UC patients (20) had extraintestinalmanifestations including arthropathy (15) primary scle-rosing cholangitis (10) and erythema nodosum (5) Allpatients were under mesalazine treatment 45 used oral or


Table 2 (a) Serum cytokines levels in each group (b) Serum cytokines levels in UC groups

(a)

UC (119899 = 20) CD (119899 = 7) HC (119899 = 15)Il-35 level (ngmL) 20336 plusmn 3821lowastlowast 45417 plusmn 21938lowastlowast 178896 plusmn 20943Il-37 level (ngmL) 19928 plusmn 3860lowastlowast 48167 plusmn 23282lowastlowast 227568 plusmn 26124

(b)

Mild UC (Mayo 3sim5) (119899 = 6) Moderate UC (Mayo 6sim10) (119899 = 7) Severe UC (Mayo 11sim12) (119899 = 7)Il-35 level (ngmL) 35826 plusmn 10395lowast 15729 plusmn 1589 11669 plusmn 1448Il-37 level (ngmL) 34697 plusmn 10583lowast 15421 plusmn 2495 11775 plusmn 1714lowastlowast

119875 lt 0001 versus HC lowast119875 lt 005 versus mild UC 119875 lt 005 versus severe UC

systemic glucocorticosteroids 5 were taking azathioprineCD patients were few Five were moderate activity and twowere severe activity Two patients had arthropathy

32 Serums IL-35 and IL-37 Levels Are Decreased in IBDPatients Serums IL-35 and IL-37 concentrations were sig-nificantly reduced in the active UC patients and active CDpatients compared with healthy controls (HC) (Table 2(a))There were also significant differences between mild UC andmoderate UC and mild UC and severe UC (119875 lt 005)(Table 2(b)) In contrast UC and CD group and moderateand mild UC group seem to be statistically meaninglessWe also assessed whether the serum cytokine levels wereassociated with the Mayo score in UC patients The resultsshowed that lower IL-35 and IL-37 levels were moderatelynegatively correlated Mayo score (119877 = minus0636 119875 lt 005119877 = minus0625 119875 lt 005 resp) (Figures 1(a) and 1(b))

33 IL-35 Expression in Colonic Mucosa from IBD PatientsIn order to determine gene and protein expressions in UCand CD patients mRNA relative expressions of Ebi3 andp35 and protein of IL-35 were quantified by qRT-PCR andWestern blot analysis IL-35 producing cells were determinedby immunohistochemistry As shown in the representativeimages of this analysis in Figures 2(a)ndash2(d) IL-35 wasexpressed in infiltrating immune cells but not in epithelialcells Normal colon tissue from healthy subjects had no IL-35 expression at all (Figures 2(e) and 2(f)) IL-35 positivecells were localized mainly in inflammatory infiltrates pre-dominantly mononuclear cell (lymphocytes) The number ofIL-35-expressing cells in inflamed colonic tissue of patientswith UC (Figures 2(a) and 2(b)) was higher than that inCD patientsrsquo tissue (Figures 2(c) and 2(d)) IBD patients hadsignificantly higher Ebi3 and p35 gene expression comparedwith healthy control group (119875 lt 00001) (Figure 3) Andthe difference between UC and CD biopsies in Ebi3 and p35mRNA expressions did not reach statistical significance (119875 =0116 119875 = 0779) Western blot analysis showed a significantupregulation of IL-35 (119875 lt 005) in inflamed mucosa ofpatients as compared to controls (Figure 4) IL-35 proteinexpression in UC biopsies was found higher than that in CDbiopsies No relationship between IL-35 mucosal expressionand the activity was observed in UC and CD patients

Seru

m IL

-35

(ng

mL)

0 5 10 15

0

200

400

600

800

1000

Y = 463321 minus 33982X

R = minus0636

Mayo score

(a)

0 5 10 15

0

200

400

600

800

1000

Y = 457084 minus 33700X

R = minus0625

Seru

m IL

-37

(ng

mL)

Mayo score

(b)

Figure 1There was an intermediate inverse correlation between theMayo score and serum IL-35 levels (a) IL-37 levels were moderatelynegatively correlated with Mayo score (b)

34 IL-37 Expression in Colonic Mucosa from IBD PatientsImmunohistochemistry showed that both immune andepithelial cells could express IL-37 (Figures 5(a)ndash5(d)) andnormal tissue had IL-37 expression though with relativelysmall amount (Figures 5(e) and 5(f)) Besides strong cyto-plasmic staining few single lamina propriamononuclear cellsshow nuclear expression of IL-37 Similarly compared with


100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

100120583m

(e)

50120583m

(f)

Figure 2 Photomicrographs of immunostaining for IL-35 in human colon from patients with UC (a and b) patients with CD (c and d)and healthy controls (e and f) No staining was found in the control group (e and f) and IL-35 was expressed in infiltrating immunecells (morphologically resembling lymphocytes) but not in epithelial cells The staining was mostly cytoplasmic and red arrows depictedimmunoreactive positive cells The number of IL-35-expressing cells in inflamed colonic tissue of patients with UC was higher than in CDpatientsrsquo tissue Original magnification (a) (c) (e) times200 (b) (d) (f) times400

control group IBDgroup had higher IL-37mRNAexpression(119875 lt 0001) whereas difference between UC and CD wasstill statistically significant (119875 lt 0001) (Figure 6) For IL-37 protein expression trend runs like IL-35 Western blotshowed that IL-37 protein expression was found to be higherin UC patients and CD patients compared to healthy subjectsand the expression levels of IL-37 protein were higher in thesamples of UC patients than that of CD samples (119875 lt 0001)(Figure 7) The mean IL-37 expression tended to be higherin severe UC samples than in mild UC samples but was notstatistically significant

4 Discussion

In this study we first demonstrated that serums IL-35 and IL-37 levels were significantly lower in active IBD patients thanhealthy controls and were moderately negatively correlatedwithMayo score inUCpatients Takahashi et al reported thatthe Treg cell frequency decreased in the active stage of UCand it correlated inversely with the disease activity [12]Theirresults suggested that a deficiency of Treg cells was associatedwith the progression of ulcerative colitis Treg cells are themain source of IL-35 which may result in the reduction


HC UC CD0

20

40

60

80

Fold

P lt 0001

P = 0116P lt 0001

Ebi3G

APDH

mRN

A

(a)

HC UC CD0

20

40

60

Fold

P lt 0001 P = 0779

P lt 0001

p35GA

PDH

mRN

A

(b)

Figure 3 Detection of Ebi3 and p35mRNA by qRT-PCR Ebi3 (a) and p35 (b) were significantly overexpressed in endoscopic specimens inboth UC patients and CD patients as compared to controls (119875 lt 0001) Ebi3 and p35mRNA expression in UC and CD biopsies did not reachstatistical significance

IL-35GAPDH

UC CD HC

(a)

UC CD HC00

02

04

06

08

10

P lt 0001

P lt 0001

P lt 0001

IL-35G

APD

H

(b)

Figure 4 Detection of IL-35 in protein extracts of endoscopicspecimens by Western blot Representative Western blot of IL-35 and GAPDH (a) proteins levels in UC and CD patients andhealthy controls A significant upregulation of IL-35 was observedin inflamed mucosa of patients as compared to controls (119875 lt 0001)and IL-35 protein expression in UC biopsies was higher than that inCD biopsies (b)

of IL-35 in peripheral blood Through immunocytochemicalstaining IL-37 protein is present mainly in the cytoplasm ofperipheral blood mononuclear cells (PBMC) and constitu-tively at low levels in normal people and can be upregulatedby inflammatory stimuli and cytokines [13] IL-37 is expected

to have the function of translocation into nucleus [14] andcan be redistributed between intracellular and extracellularPerhaps IL-37 transferring to intracellular is the reason whythe content is down in serumThe study shows that decreasedserums IL-35 and IL-37 levels may represent insufficient anti-inflammatory activity in vivo and hold promise as novelbiomarkers for monitoring disease activity in UC

We have characterized the mucosal expressions of IL-35 and IL-37 in patients with IBD The overexpressionand enhanced production of IL-35 and IL-37 in colonicmucosa may play a role in the inflammatory process ofIBD Treg cells are highly infiltrated in the lamina propria(LP) of inflamed areas of UC colon compared to normalcolon [15] Treg cells induced the generation of inducedregulatory T 35 cells (iTR 35 cells) in an IL-35- and IL-10-dependent manner in vitro and induced their generation invivo under inflammatory conditions in intestines infectedwith Trichuris muris So we think iTR 35 cells are increasedto produce more IL-35 to inhibit effective T cell (Teff cell)proliferation and suppress Th17 development Maybe theIL-35-producing B cells also participate the production ofIL-35 for Shen et al suggested that during experimentalautoimmune encephalomyelitis (EAE) CD138 (+) plasmacells were also the main source of B-cell-derived IL-35 andIL-10 [6] In the gut constitutive epithelial expression ofanti-inflammatory immune mediators like IL-37 might bemandatory to maintain the homeostasis of the local immuneresponse against commensal bacteria The protein is inducedby toll-like receptor (TLR) agonists in monocytes and isexpressed in tissues frompatients with inflammatory diseases[16 17] And the production of IL-37 by epithelial cellsneutrophils and monocytes can form a positive feedbackto promote more We speculate that the increased IL-35and IL-37 which are delivered by infiltrating immune cellscounteract mucosal inflammation in IBD The UC grouppossessed the highest expression of IL-35 and IL-37 in colonic


100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

(e) (f)

Figure 5 Photomicrographs of immunostaining for IL-37 in patients with UC (a and b) patients with CD (c and d) and control subjects (eand f) IL-37 was expressed by both immune and epithelial cells (andashd) and normal tissue had IL-37 expression though with relatively smallamount (e and f) Besides strong cytoplasmic staining few single lamina propria mononuclear cells showed nuclear expression of IL-37

tissue followed by CD group and HC group expressedthe lowest The lower anti-inflammatory cytokines in CDmay explain why CD is a more chronic and continueddisease However there was no relationship between IL-35and IL-37 mucosal expressions and the activity in UC or CDpatients It is possible that at the beginning of inflammatorydisease a large number of the immune-suppressing cytokineswere stimulated to produce to limit inflammation in theaffected colon Despite their increased frequency and potentsuppressor activity in vitro they fail to reverse the diseaseprocess Unlike IL-37 the mRNA levels of Ebi3 and p35did not show differences between UC and CD We shouldconsider the fact that IL-27 and IL-35 shared the 120573-chain Ebi3whereas IL-12 and IL-35 shared the p35 a-chain

The mechanisms of IL-35 and IL-37 are not clear untilnow IL-35 is involved in inflammatory diseases in thenervous system alimentary system bone and joint systemand respiratory system Zandian et al demonstrated thatIL-35 had an inhibitory effect against demyelination bypreventing the development of autoaggressive T cells [18]Kochetkova et al suggested that exogenous IL-35 couldsuppress the activity of CD4+T cells and Th1 and Th17 cellsand inhibit the inflammation of collagen-induced arthritis[19] Meanwhile it was indicated that IL-35 could help therespiratory system recover from inflammation [20] Wirtz etal recently confirmed that IL-35 could significantly suppressTh1 and Th17 cellsrsquo proliferation reduce the development ofexperimental colitis and protect the intestine from immune


HC UC CD0

10

20

30

40

P lt 0001 P = 0008

P = 0003Fold

IL-37GA

PDH

mRN

A

Figure 6 IL-37 gene expression in colonic mucosa from IBDpatients A significant increase in IL-37 mRNA was shown ininflamed mucosa of patients as compared to controls

HC UCCD

IL-37

GAPDH

(a)

HC CD UC00

05

10

15

20

P lt 0001

P lt 0001

P lt 0001

IL-37G

APD

H

(b)

Figure 7 Detection of IL-37 in protein extracts of endoscopicspecimens by Western blot Representative Western blot of IL-37and GAPDH (a) proteins levels in UC and CD patients and healthycontrols IL-37 protein expression was found to be higher in UCpatients and CD patients compared to healthy subjects and theexpression levels of IL-37 protein were higher in the samples of UCpatients than that of CD samples (b)

responses in mice [21] One subunit of IL-35 Ebi3 is widelyexpressed in EBV-transformed B-lymphocytes and tissuessuch as tonsil and spleen [22] Ebi3 could negatively regulateIL-17 IL-22 and Th17 transcription factor ROR120574t and exertprotective immunity against inflammation [23] The subunitof IL-12 p35 could lead to the progression of Herpes stromalkeratitis (HSK) in mice which is IL-12p40 independent [24]The two subunits of IL-35 do have their own ability to regulateimmunity and the process of inflammation When they

combine together to form the heterodimer the p35 subunitmay act as a ligand and the other subunit EBI3 may mainlyexert its immunological function [25] So far the signalingpathway of IL-35 is not clear yet Meanwhile the researchconfirmed that IL-35 signaled through a unique heterodimerof receptor chains IL12R1205732 and gp130 or homodimers of eachchain [26] Signaling through the IL-35 receptor requiredthe transcription factors STAT1 and STAT4 which formeda unique heterodimer that bounded to distinct sites in thepromoters of the genes encoding the IL-12 subunits p35 andEbi3 IL-35 can directly suppress Teff cell proliferation con-vert naive T cells into IL-35-producing iTr35 cells suppressTh17 development and mediate IL-10 generation SimilarlyIL-37 is a cytokine for inflammation autoimmunity andother immunological disorders The IL-37 protein is highlyexpressed in synovial cells of patients with rheumatoid arthri-tis but expressed at low levels in healthy human synovial cells[5 27] IL-37 expression was also significantly increased inthe skin lesions of patients with psoriasis and inmacrophagesof Crohnrsquos disease lesions [28] IL-37 is synthesized as aproprotein which after stimulation is processed to itsmatureform [28] Lipopolysaccharide (LPS) together with otherinflammatory stimuli and cytokines activates caspase-1 andis considered to be the major cleaving enzyme responsiblefor maturation of IL-1 family precursors [16] With broad-spectrum function in antibacterial antiviral neutralizingendotoxins and antitumor and immune regulation IL-37 cankill the microorganism in general And its mechanism ismainly by changing the permeability of bacterial cells It alsohas the ability to raise the production of several cytokinessuch as IL-8 to expand acquired immune function [29]Studies of mouse models have reached the result that IL-37 downregulates inflammation [3] TLR tumour necrosisfactor (TNF) and other cytokines can induce the productionof inflammatory cytokines Nold et al reported that IL-37attenuated the abovementioned process thus exerting anti-inflammatory effects [27] Besides Liu et al proved that IL-37 exerted a significant inhibition on TNF-120572-induced IP-10expression [30] In the inflamed mucosa of IBD patientsT cell activation as well as dendritic cells (DCs) activa-tion can be inhibited by epithelial cell-derived IL-37 Thepossible mechanism may be that IL-37 reduces the surfaceexpressions of the costimulatory molecule CD86 (B7-2) andmajor histocompatibility complex (MHC) II on DCs IL-37b mRNA expression induced by TNF-120572 was mediated bythe activation of MAPK and PI3K and transcription factorsNF-kB and AP-1 Conversely these signalling moleculesare major mediators of the induction of proinflammatoryresponses in the inflamed mucosa Thus it became clear thatin the inflamed mucosa of IBD patients a negative feedbackinhibitor of inflammatory responses (the induction of IL37b)and proinflammatory responses was induced via coupledsignalling pathways [31]

5 Conclusion

IL-35 and IL-37 are brand new potential therapeuticcytokines for IBD Our experiment group (namely UC group


and CD group) includes 27 cases and large-scale testingneeds to be performed Thus further mechanism studies onthe roles of IL-35 and IL-37 should be performed to make itavailable and useful in the future

Abbreviations

IL-35 Interleukin 35IL-37 Interleukin 37CD Crohnrsquos diseaseUC Ulcerative colitisIBD Inflammatory bowel diseasePBMC Peripheral blood mononuclear cellsqRT-PCR Quantitative real-time

polymerase-chain reactioniTR35 Induced regulatory 35 cellTeff Effective T cellTLR Toll-like receptorDCs Dendritic cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Yanmei Li and YananWang equally contributed to the paper

References

[1] D K Podolsky ldquoInflammatory bowel diseaserdquoTheNewEnglandJournal of Medicine vol 347 no 6 pp 417ndash429 2002

[2] T TMacDonald andGMonteleone ldquoImmunity inflammationand allergy in the gutrdquo Science vol 307 no 5717 pp 1920ndash19252005

[3] K J Maloy and F Powrie ldquoIntestinal homeostasis and itsbreakdown in inflammatory bowel diseaserdquoNature vol 474 no7351 pp 298ndash306 2011

[4] L W Collison C J Workman T T Kuo et al ldquoThe inhibitorycytokine IL-35 contributes to regulatory T-cell functionrdquoNature vol 450 no 7169 pp 566ndash569 2007

[5] V Chaturvedi L W Collison C S Guy C J Workman and DA A Vignali ldquoCutting edge human regulatory T cells requireIL-35 to mediate suppression and infectious tolerancerdquo Journalof Immunology vol 186 no 12 pp 6661ndash6666 2011

[6] P Shen T Roch V Lampropoulou et al ldquoIL-35-producing Bcells are critical regulators of immunity during autoimmuneand infectious diseasesrdquoNature vol 507 no 7492 pp 366ndash3702014

[7] X Li J Mai A Virtue et al ldquoIL-35 is a novel responsive anti-inflammatory cytokinemdasha new system of categorizing anti-inflammatory cytokinesrdquo PLoS ONE vol 7 no 3 Article IDe33628 2012

[8] Y K Lee and S K Mazmanian ldquoHas the microbiota played acritical role in the evolution of the adaptive immune systemrdquoScience vol 330 no 6012 pp 1768ndash1773 2010

[9] P Chen and S Fang ldquoThe expression of human antimicrobialpeptide LL-37 in the human nasal mucosardquo The AmericanJournal of Rhinology vol 18 no 6 pp 381ndash385 2004

[10] G DrsquoHaens W J Sandborn B G Feagan et al ldquoA review ofactivity indices and efficacy end points for clinical trials of med-ical therapy in adults with ulcerative colitisrdquo Gastroenterologyvol 132 no 2 pp 763ndash786 2007

[11] R F Harvey and J M Bradshaw ldquoA simple index of Crohnrsquos-disease activityrdquoThe Lancet vol 1 no 8167 p 514 1980

[12] M Takahashi K Nakamura K Honda et al ldquoAn inverse cor-relation of human peripheral blood regulatory T cell frequencywith the disease activity of ulcerative colitisrdquo Digestive Diseasesand Sciences vol 51 no 4 pp 677ndash686 2006

[13] S Kumar C R Hanning M R Brigham-Burke et alldquoInterleukin-1F7b (IL-1H4IL-1F7) is processed by caspase-1and mature IL-1F7b binds to the IL-18 receptor but does notinduce IFN-120574 productionrdquo Cytokine vol 18 no 2 pp 61ndash712002

[14] P Bufler F Gamboni-Robertson T Azam S Kim and C ADinarello ldquoInterleukin-1 homologues IL-1F7b and IL-18 containfunctional mRNA instability elements within the coding regionresponsive to lipopolysacchariderdquo Biochemical Journal vol 381no 2 pp 503ndash510 2004

[15] Q T YuM Saruta A Avanesyan P R Fleshner A H Banhamand K A Papadakis ldquoExpression and functional characteriza-tion of FOXP3+CD4 + regulatory T cells in ulcerative colitisrdquoInflammatory Bowel Diseases vol 13 no 2 pp 191ndash199 2007

[16] P Bufler T Azam F Gamboni-Robertson et al ldquoA complex ofthe IL-1 homologue IL-1F7b and IL-18-binding protein reducesIL-18 activityrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 99 no 21 pp 13723ndash137282002

[17] S Sharma N Kulk M F Nold et al ldquoThe IL-1 familymember 7b translocates to the nucleus and down-regulatesproinflammatory cytokinesrdquo Journal of Immunology vol 180no 8 pp 5477ndash5482 2008

[18] M Zandian K R Mott S J Allen O Dumitrascu J Z Kuoand H Ghiasi ldquoUse of cytokine immunotherapy to block CNSdemyelination induced by a recombinant HSV-1 expressing IL-2rdquo Gene Therapy vol 18 no 7 pp 734ndash742 2011

[19] I Kochetkova S Golden K Holderness G Callis and DW Pascual ldquoIL-35 stimulation of CD39+ regulatory T cellsconfers protection against collagen II-induced arthritis via theproduction of IL-10rdquo Journal of Immunology vol 184 no 12 pp7144ndash7153 2010

[20] G S Whitehead R H Wilson K Nakano L H BurchH Nakano and D N Cook ldquoIL-35 production by induciblecostimulator (ICOS)-positive regulatory T cells reverses estab-lished IL-17-dependent allergic airways diseaserdquo Journal ofAllergy and Clinical Immunology vol 129 no 1 pp 207ndash2152012

[21] S Wirtz U Billmeier T McHedlidze R S Blumberg andM FNeurath ldquoInterleukin-35 mediates mucosal immune responsesthat protect against T-cell-dependent colitisrdquo Gastroenterologyvol 141 no 5 pp 1875ndash1886 2011

[22] O Devergne M Birkenbach and E Kieff ldquoEpstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form anovel heterodimeric hematopoietinrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 94 no22 pp 12041ndash12046 1997

[23] J Yang M Yang T M Htut et al ldquoEpstein-Barr virus-inducedgene 3 negatively regulates IL-17 IL-22 and ROR120574trdquo EuropeanJournal of Immunology vol 38 no 5 pp 1204ndash1214 2008

[24] G M Frank S J Divito D M Maker M Xu and R LHendricks ldquoA novel P40-independent function of IL-12P35 is


required for progression and maintenance of herpes stromalkeratitisrdquo Investigative Ophthalmology and Visual Science vol51 no 7 pp 3591ndash3598 2010

[25] S Ye J Wu L Zhou Z Lv H Xie and S Zheng ldquoInterleukin-35 the future of hyperimmune -related diseasesrdquo Journal ofInterferon and Cytokine Research vol 33 no 6 pp 285ndash2912013

[26] LWCollisonGMDelgoffe C SGuy et al ldquoThe compositionand signaling of the IL-35 receptor are unconventionalrdquo NatureImmunology vol 13 no 3 pp 290ndash299 2012

[27] M F Nold C A Nold-Petry J A Zepp B E Palmer P Buflerand C A Dinarello ldquoIL-37 is a fundamental inhibitor of innateimmunityrdquo Nature Immunology vol 11 no 11 pp 1014ndash10222010

[28] D Boraschi D Lucchesi S Hainzl et al ldquoIL-37 A new anti-inflammatory cytokine of the IL-1 familyrdquo European CytokineNetwork vol 22 no 3 pp 127ndash147 2011

[29] K de Smet and R Contreras ldquoHuman antimicrobial peptidesdefensins cathelicidins andhistatinsrdquoBiotechnology Letters vol27 no 18 pp 1337ndash1347 2005

[30] M Liu S Guo J M Hibbert et al ldquoCXCL10IP-10 in infectiousdiseases pathogenesis and potential therapeutic implicationsrdquoCytokine and Growth Factor Reviews vol 22 no 3 pp 121ndash1302011

[31] H Imaeda K Takahashi T Fujimoto et al ldquoEpithelial expres-sion of interleukin-37b in inflammatory bowel diseaserdquo Clinicaland Experimental Immunology vol 172 no 3 pp 410ndash416 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


2 Materials and Methods

21 Subjects A total of 27 patients with definitive diagnosisof IBD were recruited at Shandong University Qilu Hospital20UC and 7CD patients were included during the periodfrom September 2013 to April 2014 Diagnosis was performedby the presence of history of abdomen pain diarrhea orblood in stool and macroscopic appearance by colonoscopyor double balloon endoscopy and biopsy compatible withIBD The following were relevant medical records genderage at diagnosis disease evolution extension extraintestinalmanifestations medical treatment and clinical course ofdisease UC activity was assessed byMayo score activity index[10] and CD activity was assessed by Crohnrsquos disease activityindex (CDAI) [11] Blood was drawn for the measurementof hemoglobin hematocrit and erythrocyte sedimentationrate (ESR) Additionally 15 noninflamed controls (medianage 48 yr 9 males6 females) were recruited among healthysubjects undergoing a colonoscopy because of screening forcolorectal cancer or polyp surveillance These subjects werefree from gastrointestinal symptoms and other inflamma-tory diseases Only subjects with both macroscopically andmicroscopically normal colonoscopy were included None ofthe healthy subjects in the study was taking any medicationsknown to affect the gastrointestinal tract or the immunesystem

22 Samples Collection A fasting blood sample was takenfrom all patients and healthy subjects It was centrifugedat 1500timesg for 20min at room temperature and the serumwas collected and stored at minus80∘C until analysis Duringendoscopy biopsies from colon or ileocecum or smallintestine were obtained from patients and healthy subjectsTwo biopsies to be used for RNA and protein assessmentwere snap-frozen in liquid nitrogen and then transferredto minus80∘C for storage until processing One biopsy wasplaced in formalin for pathology and immunohistochemicalstaining The study was performed after receiving writteninformed consent from all study subjects and the protocolwas approved by the Regional Ethical Review Board at QiluHospital Shandong University

23 Enzyme-Linked ImmunosorbentAssay Serums IL-35 andIL-37 were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Bio-Swamp)All cytokines assays were performed in duplicate and inaccordance with the manufacturersrsquo protocols

24 Immunohistochemistry Formalin-fixed and paraffin-embedded 4 120583m thick tissue slices were dewaxed and rehy-drated before antigen retrieval Microwave antigen retrievalmethod was then preformed with the slides immersed inEDTA antigen retrieval solution (ph 90) for 15minutes Afterthat 3 hydrogen peroxide (H

2O2) was added on the slides

to inhibit the endogenous peroxidase activity Nonspecificbinding was blocked by incubation with 10 normal goatserum in 37∘C pH75 for 30min Subsequently mouse anti-human IL-35 monoclonal antibody (Imgenex USA) at a

1 200 dilution and mouse anti-human IL-37 monoclonalantibody (Abcam USA) at a 1 250 dilution were appliedrespectively to the sections that were latter incubated at 4∘Covernight On the second day biotinylated antibody andstreptavidin-peroxidase reagent (Zhongshan Biotech China)were successively applied for 30min each at 37∘C Finally31015840-diaminobenzidine tetrahydrochloride (DAB) was used forvisualization and hematoxylin was added to counterstainSamples were viewed with Olympus IX81 microscope andimages were produced using DP Controller 121108 All ofthe slides were independently analyzed by two pathologists

25 RNA Isolation and Quantitative Real-Time PCR RNAwas extracted using Trizol Reagent (Takara Japan) follow-ing the manufacturerrsquos guidelines First-strand cDNA wassynthesized by Realtime PCR Master Mix Kit (TOYOBOJapan) in a volume of 10 120583L For quantitative real-time PCR(qRT-PCR) the LightCycler 40 instrument (Roche AppliedScience Germany) and the SYBR Green Realtime PCRMaster Mix Kit (TOYOBO Japan) were used according tothe protocol provided by the manufacturer The followingprimers were used Ebi3 forward 51015840-GCA GCA GAC GCCAAC GT-31015840 reverse 51015840-CCA TGG AGA ACA GCT GGACAT-31015840 p35 forward 51015840-CCT TCA CCA CTC CCA AAAC-31015840 reverse 51015840-TGT CTG GCC TTC TGG AGC AT-31015840IL-37 forward 51015840-GCT CAG GTG GGC TCC TGG AA-31015840reverse 51015840-GCT GAC CTC ACT GGG GCT CA-31015840 Humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH) wasused as internal control from parallel samples because thereference gene was stably expressed and its primers wereforward 51015840-GGT GGT CTC CTC TGA CTT CAA CAG-31015840and reverse 51015840-GTT GTT GTA GCC AAA TTC GTT GT-31015840 Melting curve analysis was used to confirm amplifica-tion specificity The quantification data were analyzed withLightCycler analysis software version 40 (Roche AppliedScienceGermany) and the relative target gene expressionwasnormalized on the basis ofGAPDH Results were expressed asan x-fold difference relative to the calibrator

26 Western Blot Analysis Frozen tissue samples were lysedin RIPA buffer (50mM Tris pH 74 150mM NaCl 1Triton X-100 1 sodium deoxycholate 01 SDS Bey-otime China) and PMSF (Beyotime China) followed bycentrifugation (12000 rpm 4∘C 20 minutes) after whichthe supernatants were stored at minus80∘C until use The pro-tein concentrations of the lysates were determined usingan enhanced BCA Protein Assay Kit (Beyotime China)Extracted proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Samplestransferred onto PVDFmembranes were treated with a 1 200dilution of mouse anti-human IL-35 monoclonal antibody(Imgenex USA) and anti-human IL-37monoclonal antibody(Abcam USA) followed by a 1 5000 dilution of sheep anti-mouse horseradish peroxidase-conjugated secondary anti-bodies (Zhongshan Biotech China) As a control rabbit anti-human GAPDH polyclonal antibody (Proteintech USA) andperoxidase-conjugated anti-rabbit IgG (H+L) (Proteintech










3 Results




(a)


(b)






Seru

m IL

-35

(ng

mL)

0 5 10 15

0

200

400

600

800

1000

Y = 463321 minus 33982X

R = minus0636

Mayo score

(a)

0 5 10 15

0

200

400

600

800

1000

Y = 457084 minus 33700X

R = minus0625

Seru

m IL

-37

(ng

mL)

Mayo score

(b)




100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

100120583m

(e)

50120583m

(f)



4 Discussion



HC UC CD0

20

40

60

80

Fold

P lt 0001

P = 0116P lt 0001

Ebi3G

APDH

mRN

A

(a)

HC UC CD0

20

40

60

Fold

P lt 0001 P = 0779

P lt 0001

p35GA

PDH

mRN

A

(b)


IL-35GAPDH

UC CD HC

(a)

UC CD HC00

02

04

06

08

10

P lt 0001

P lt 0001

P lt 0001

IL-35G

APD

H

(b)






100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

(e) (f)





HC UC CD0

10

20

30

40

P lt 0001 P = 0008

P = 0003Fold

IL-37GA

PDH

mRN

A


HC UCCD

IL-37

GAPDH

(a)

HC CD UC00

05

10

15

20

P lt 0001

P lt 0001

P lt 0001

IL-37G

APD

H

(b)




5 Conclusion




Abbreviations







References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























3 Results




(a)


(b)






Seru

m IL

-35

(ng

mL)

0 5 10 15

0

200

400

600

800

1000

Y = 463321 minus 33982X

R = minus0636

Mayo score

(a)

0 5 10 15

0

200

400

600

800

1000

Y = 457084 minus 33700X

R = minus0625

Seru

m IL

-37

(ng

mL)

Mayo score

(b)




100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

100120583m

(e)

50120583m

(f)



4 Discussion



HC UC CD0

20

40

60

80

Fold

P lt 0001

P = 0116P lt 0001

Ebi3G

APDH

mRN

A

(a)

HC UC CD0

20

40

60

Fold

P lt 0001 P = 0779

P lt 0001

p35GA

PDH

mRN

A

(b)


IL-35GAPDH

UC CD HC

(a)

UC CD HC00

02

04

06

08

10

P lt 0001

P lt 0001

P lt 0001

IL-35G

APD

H

(b)






100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

(e) (f)





HC UC CD0

10

20

30

40

P lt 0001 P = 0008

P = 0003Fold

IL-37GA

PDH

mRN

A


HC UCCD

IL-37

GAPDH

(a)

HC CD UC00

05

10

15

20

P lt 0001

P lt 0001

P lt 0001

IL-37G

APD

H

(b)




5 Conclusion




Abbreviations







References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


(b)






Seru

m IL

-35

(ng

mL)

0 5 10 15

0

200

400

600

800

1000

Y = 463321 minus 33982X

R = minus0636

Mayo score

(a)

0 5 10 15

0

200

400

600

800

1000

Y = 457084 minus 33700X

R = minus0625

Seru

m IL

-37

(ng

mL)

Mayo score

(b)




100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

100120583m

(e)

50120583m

(f)



4 Discussion



HC UC CD0

20

40

60

80

Fold

P lt 0001

P = 0116P lt 0001

Ebi3G

APDH

mRN

A

(a)

HC UC CD0

20

40

60

Fold

P lt 0001 P = 0779

P lt 0001

p35GA

PDH

mRN

A

(b)


IL-35GAPDH

UC CD HC

(a)

UC CD HC00

02

04

06

08

10

P lt 0001

P lt 0001

P lt 0001

IL-35G

APD

H

(b)






100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

(e) (f)





HC UC CD0

10

20

30

40

P lt 0001 P = 0008

P = 0003Fold

IL-37GA

PDH

mRN

A


HC UCCD

IL-37

GAPDH

(a)

HC CD UC00

05

10

15

20

P lt 0001

P lt 0001

P lt 0001

IL-37G

APD

H

(b)




5 Conclusion




Abbreviations







References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

100120583m

(e)

50120583m

(f)



4 Discussion



HC UC CD0

20

40

60

80

Fold

P lt 0001

P = 0116P lt 0001

Ebi3G

APDH

mRN

A

(a)

HC UC CD0

20

40

60

Fold

P lt 0001 P = 0779

P lt 0001

p35GA

PDH

mRN

A

(b)


IL-35GAPDH

UC CD HC

(a)

UC CD HC00

02

04

06

08

10

P lt 0001

P lt 0001

P lt 0001

IL-35G

APD

H

(b)






100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

(e) (f)





HC UC CD0

10

20

30

40

P lt 0001 P = 0008

P = 0003Fold

IL-37GA

PDH

mRN

A


HC UCCD

IL-37

GAPDH

(a)

HC CD UC00

05

10

15

20

P lt 0001

P lt 0001

P lt 0001

IL-37G

APD

H

(b)




5 Conclusion




Abbreviations







References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HC UC CD0

20

40

60

80

Fold

P lt 0001

P = 0116P lt 0001

Ebi3G

APDH

mRN

A

(a)

HC UC CD0

20

40

60

Fold

P lt 0001 P = 0779

P lt 0001

p35GA

PDH

mRN

A

(b)


IL-35GAPDH

UC CD HC

(a)

UC CD HC00

02

04

06

08

10

P lt 0001

P lt 0001

P lt 0001

IL-35G

APD

H

(b)






100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

(e) (f)





HC UC CD0

10

20

30

40

P lt 0001 P = 0008

P = 0003Fold

IL-37GA

PDH

mRN

A


HC UCCD

IL-37

GAPDH

(a)

HC CD UC00

05

10

15

20

P lt 0001

P lt 0001

P lt 0001

IL-37G

APD

H

(b)




5 Conclusion




Abbreviations







References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100120583m

(a)

50120583m

(b)

100120583m

(c)

50120583m

(d)

(e) (f)





HC UC CD0

10

20

30

40

P lt 0001 P = 0008

P = 0003Fold

IL-37GA

PDH

mRN

A


HC UCCD

IL-37

GAPDH

(a)

HC CD UC00

05

10

15

20

P lt 0001

P lt 0001

P lt 0001

IL-37G

APD

H

(b)




5 Conclusion




Abbreviations







References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HC UC CD0

10

20

30

40

P lt 0001 P = 0008

P = 0003Fold

IL-37GA

PDH

mRN

A


HC UCCD

IL-37

GAPDH

(a)

HC CD UC00

05

10

15

20

P lt 0001

P lt 0001

P lt 0001

IL-37G

APD

H

(b)




5 Conclusion




Abbreviations







References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Abbreviations







References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article the possible role of the novel cytokines...

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